Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Rescue of the tail defect of Brachyury mice David Stott, ~'2 Andreas Kispert, and Bernhard G. Herrmann Max-Planck-Institut fiir Entwicklungsbiologie, 7400 Tfibingen, Germany The mouse Brachyury (T) gene is required for normal development of axial structures. Embryos homozygous for the T mutation show severe deficiencies in mesoderm formation. They lack the notochord and allantois, have abnormal somites, and die at -10 days postcoitum probably as a result of the allantois defect. Mice heterozygous for the T mutation exhibit a variable short-tailed phenotype. The T gene has been cloned and shown to be expressed in the tissues most strongly affected by the mutation. In this paper, we show that a single-copy transgene representing the wild-type T allele is able to rescue the T-associated tail phenotype. In addition, we show that increasing dosage of the T gene in T~/+ mice causes an increased extension of the axis. These data show the correlation of the level of T product with the extension of the anteroposterior axis, directly demonstrating the involvement of the T product in this process. [Key Words: Brachyury; mouse development; phenotypic rescue; axis; mouse embryo; tail interaction factor] Received September 24, 1992; revised version accepted November 17, 1992. Identification and cloning of genes encoding products re- about this axis. The inner layer of cells, referred to as the quired for essential developmental processes has proved primitive ectoderm or epiblast, is thought to give rise to to be a productive route toward understanding the mech- all structures of the embryo proper (Gardner and Papaio- anisms underlying cellular differentiation and pattern annou 1975; Gardner and Rossant 1979; Gardner et al. formation during animal development. In the case of the 1985; Beddington et al. 1989}, whereas the outer layer, mammalian embryo, long life cycles, intrauterine devel- the visceral endoderm, appears essentially not to con- opment, and genome size mitigate against genetic tribute to the embryo (Lawson et al. 1991). This cylinder screens to identify large numbers of loci encoding differ- is surrounded by the parietal yolk sac, composed of pa- ent functional components involved in particular events. rietal endoderm and trophoblast cells, which interacts An alternative approach is to work from the product of a directly with maternal tissues. The first indication of the single locus shown to be required for an event in an anteroposterior axis of the developing embryo is the ap- attempt to understand its role in development and, sub- pearance of the primitive streak, which forms initially sequently, identify interacting components. We have about halfway along the length of the cylinder and ex- chosen to study the action of the product of a gene, tends, in the space of -8 hr, to the distal tip. The streak Brachyury or T, which is known from the phenotype of is a region of the egg cylinder where cells leave the inner mutant strains of mice to be required for the processes of layer--referred to as the epiblast or primitive ectoderm--to mesoderm formation and anteroposterior axis elabora- migrate away between the two layers of the cylinder (Na- tion in the mammalian embryo (Chesley 1935; Glueck- katsuji et al. 1986) eventually forming paraxial meso- sohn-Schoenheimer 1944; Spiegelman 1976). This gene derm and other mesodermal cell types. The appearance has been cloned (Herrmann et al. 1990) and shown to be of the primitive streak defines the posterior end of the expressed in the embryonic tissues most strongly af- axis, which is initially U-shaped, running from the prim- fected by the mutation (Wilkinson et al. 1990; Herrmann itive streak around the distal tip to the future anterior of 1991). The availability of several mutant T alleles plus the embryo on the other side of the cylinder. After ex- the cloned gene sequences allow access to the study of tension of the primitive streak, a structure forms at the the function of this gene during axis formation. distal tip of the egg cylinder, referred to as the archen- Before the appearance of the primitive streak, the teron or node. At the node, cells also emerge from the mammalian embryo consists of a two-layered cylinder inner layer but, instead of migrating between the two possessing obvious proximal distal polarity with refer- pre-existing layers, remain at the midline and intercalate ence to maternal tissues, but apparent radial symmetry into the visceral endoderm layer to form axial meso- derm, the notochordal plate, and subsequently noto- chord (Jurand 1974; Lawson et al. 1991). ICorresponding author. 2present address: Animal Molecular Genetics Group,Department of Bi- In T mutant embryos, all mesodermal cell types are ological Sciences, University of Warwick, Coventry, CV4 7AL, U.K. defective. Whereas mice heterozygous for a deletion at GENES & DEVELOPMENT7:197-203 91993 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/93 $3.00 197 Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Stott et al. the Brachyury locus display a viable short-tailed pheno- and 5 kb of 3' nontranscribed flanking sequence (Fig. 1), type, embryos homozygous for the null allele, T, form was excised from cosmid c2.190 [isolated from a 129 insufficient axial and paraxial mesoderm resulting in the strain genomic library (Herrmann et al. 1990)] using ClaI failure of the extension of the axis during early organo- and injected into fertilized eggs derived from matings of genesis and subsequent embryonic mortality, probably CBA x C57B1/6 F: mice. This fragment can be detected owing to inability to form the allantois (Chesley 1935; either by polymerase chain reaction (PCR) using primers Gluecksohn-Schoenheimer 1938; Grueneberg 1958; that amplify 220 bp of a 352-bp region of vector se- Yanagisawa et al. 1981). Abnormalities of the neural quences present at the 5' end of the transgene fragment tube are probably the result of secondary effects resulting (Figs. 1 and 3) or by genomic Southern blotting using a from the notochord deficiency. Analysis of the severity probe (190R10RS) that detects a TaqI polymorphism of the phenotype resulting from different T alleles has present between strain 129 and both CBA and C57B1/6 led to the proposal that continuing axial elaboration re- mice (Fig. 2a; see Materials and methods). Of a total of 55 quires increasingly high doses of the T product (MacMur- live-born offspring produced in three experiments, only 1 ray and Shin 1988; Yanagisawa 1990). In addition, the (designated 118.9) carried the transgene fragment, which recent observation that expression of the Xenopus ho- was present as a single copy (Fig. 2a) and is referred to as molog of Brachyury is an immediate early response to a TG T. This animal was phenotypically normal. Careful signal that mimics mesoderm induction in vitro, sup- examination revealed no difference in tail phenotype ports the proposal that the Brachyury product plays a compared with nontransgenic littermates. As 1 in 55 rep- key role in the establishment and elaboration of the me- resents an unusually low rate of transgenesis (which is soderm and, hence, of the anteroposterior axis (Smith et usually between 15% and 35% of live-born animals in al. 1991). our hands), a further experiment was performed in which In this work we show that a single-copy transgene in- injected embryos were recovered after 9 days postreim- cluding all transcribed sequences of the Brachyury gene, plantation, at the four- to eight-somite stage. Of 21 em- is sufficient to completely rescue the short tailed pheno- bryos recovered, 7 tested positive for the presence of the type of mice heterozygous for the T mutation, establish- transgene using PCR. This result suggests that the pres- ing beyond doubt that the cloned gene responsible for the ence of extra copies of the T gene may be deleterious to observed phenotype. Furthermore, by manipulating the successful development to term, but as yet no obvious copy number of the T gene in transgenic mice, we show phenotype has been detected. a direct correlation between levels of T product and axial extension. Complementation of the tail phenotype of Brachyury mutants Results To test the ability of TG T to substitute for the wild-type Generation of transgenic mice carrying an extra copy of the mouse T gene Brachyury allele, founder mouse 118.9 was crossed to mice that carried either the T mutant version of chro- Previously, we have cloned a gene which, based on the mosome 17 or the T Curtailed (7~) allele. Like T, 7~ was alteration of its transcription unit in the T w~s allele and X-ray induced and exhibits an embryonic lethal homozy- its expression in tissues affected by the mutation, most gous phenotype; but, in contrast to the variable short- likely represents the mouse T gene (Herrmann et al. tailed phenotype of T/+ animals, heterozygous 7~ mice 1990; Wilkinson et al. 1990). To test whether the prod- invariably completely lack tails and exhibit relatively uct of this RNA is capable of rescuing the phenotype of high mortality between birth and weaning (Searle 1966). Brachyury mice, we first characterized the structure of Both alleles result in a lethal homozygous phenotype, in the genomic locus corresponding to the eDNA clone which posterior regions of the embryo fail to form (see pme75 using a combination of sequencing of genomic introductory section). Sequencing of the transcribed re- subclones to identify the number and positions of exons gions of Brachyury genomic clones isolated from a li- and RNase protection and primer extension analysis to brary produced from 7-~/+ DNA revealed a 19-bp dele- ascertain the transcriptional start site (data not shown).